Activation and aggregation of human platelets leads to the formation of blood clots (thrombi). It is well established that the binding of fibrinogen to specific receptors on platelets is essential for platelet aggregation. Unstimulated platelets do not bind fibrinogen and do not aggregate during circulation. When platelets are stimulated by certain physiological agonists, such as ADP, thrombin, etc., the fibrinogen receptors associated with the glycoprotein IIb/IIIa complex on the platelet become exposed, resulting in fibrinogen binding leading to platelet aggregation.
Aggregen (M.sub.r =100 kDa) is a putative ADP-receptor on the platelet surface. It has been shown to be completely cleaved during thrombin- and plasmin-induced platelet aggregation. The binding of thrombin and plasmin to their receptors on the platelet surface is a important requirement for these plasma proteases to elicit aggregen proteolysis and platelet aggregation.
Thrombin- and plasmin-induced platelet aggregation and cleavage of aggregen are indirectly mediated by intracellularly activated calpain expressed on the platelet surface. Thrombin- and plasmin-induced platelet aggregation are inhibited by cysteine protease inhibitors, including high molecular weight kininogen. The latter is also an inhibitor of platelet calpain.
Matsueda et al., Chem. Lett. 1857-1860 (1988) disclose synthetic peptides containing a S-(3-nitro-2-pyridinesulfonyl)-cysteine residue which have activity as inhibitors of cathepsin B. The latter is an intracellular proteolytic enzyme which belongs to the group of closely related thiol proteases including cathepsin H and L.
The heavy chain of high molecular weight kininogen contains three repeating units having mutual sequence homology, designated D1, D2, and D3. Of these three repeats, D2 and D3 contain the pentapeptide Gln-Val-Val-Ala-Gly ("QVVAG"). D2 and D3 are inhibitors of cysteine proteases, with D2 being effective in inhibiting calpain, Salvesen et al., Biochem. J. 234, 429-434 (1986); Muller-Esterl Atemw.-Lungenkrkh. Jahrgang 14, 1.Suppl.-Heft S11-S22 (1988). Although the inhibitory activity of high molecular weight kininogen and other cysteine protease inhibitors have been attributed to the QVVAG sequence, it has never been demonstrated that the pentapeptide alone has inhibitory activity against calpain. Teno et al., Int. J. Peptide Protein Res. 30, 93-98 (1987) report weak activity of the QVAAG pentapeptide in inhibiting the thiol protease papain.
Reocclusion of coronary arteries is a frequent complication following thrombolytic therapy. It has been postulated that reocclusion is due to plasmin-induced activation of platelets. High concentrations of plasmin, such as might occur in therapeutic thrombolysis, are known to cause platelet aggregation.
Coronary artery restenosis following angioplasty has been linked to platelet activation by protease agonists. Restenosis may be initiated by thrombin-stimulated release of growth factors from platelets.
What is needed is a method of inhibiting stimulation and aggregation of platelets by protease agonists, specifically a method of inhibiting stimulation of platelets by inhibiting the action of platelet calpain in facilitating thrombin- and plasmin-induced platelet aggregation.